Significance of pSmad2/3 and Smad4 in hepatitis C virus‐related liver fibrosis and hepatocellular carcinoma

Chronic hepatitis C (CHC) is a major public health problem, especially in Egypt. Risk of hepatocellular carcinoma (HCC) development increases as hepatitis C virus (HCV)‐related liver diseases progress. Smads act as substrates for the transforming growth factor‐beta (TGF‐β) family of receptors. This study aims to assess hepatic expression of pSmad2/3 and Smad4 in CHC with different stages of fibrosis and grades of necro‐inflammation as well as in HCC on top of CHC. This study was done on 33 core liver biopsies from patients with CHC (15 with early fibrosis and 18 with late fibrosis), 15 liver specimens from HCC cases on top of CHC, as well as five normal controls. pSmad2/3 and Smad4 show more immunopositivity, higher percentage of positive hepatocytes and stronger staining intensity in CHC with late fibrosis compared to early fibrosis. pSmad2/3 shows increase of the previous parameters in CHC with high grade activity than those with low activity. Smad4 shows increase of the previous parameters in HCC compared to CHC cases. pSmad2/3 and Smad4 can be used as diagnostic and/or prognostic markers for progression of HCV‐related fibrosis to cirrhosis and further progression to HCC.     

Imbalance of receptor-regulated and inhibitory Smads in lung fibroblasts from bleomycin-exposed rats

Transforming growth factor (TGF)-beta plays a central role in lung fibrosis, stimulating extracellular matrix deposition. Intracellular signaling of TGF-beta is mediated by Smad proteins. We questioned whether the expression and activation of Smads would be altered in lung fibroblasts from rats exposed to bleomycin, an agent used to provoke an experimental model of lung fibrosis. Fibroblasts were isolated from rat lungs 14 d after intratracheal instillation of bleomycin (BLF) or saline (NLF), and cell cultures established. Whole cell lysates were obtained at baseline, and after stimulation with TGF-beta1 (10 ng/ml). Western blot analysis was performed to measure levels of phosphorylated Smad3 (p-Smad3) and Smad7. Real-time PCR was used to determine changes in Smad7 mRNA after TGF-beta stimulation. We found increased baseline levels of p-Smad3 in BLF versus NLF (P < 0.05). In contrast, baseline levels of Smad7 were comparable. The ratio of stimulatory to inhibitory Smads was increased in BLF compared with NLF (P < 0.05). After stimulation with TGF-beta, levels of p-Smad3 were increased in both groups, with maximal responses at 30 min (P < 0.01). While Smad7 mRNA levels were significantly upregulated (at 1 h) after TGF-beta in both groups, the increase in Smad7 protein was significant in NLF only. We conclude there is sustained activation of Smad signaling in lung fibroblasts isolated from bleomycin-exposed rats, with an imbalance between the levels of p-Smad3 and Smad7. Insufficient levels of the inhibitory Smad7 at baseline, and inadequate response to TGF-beta, may contribute to the fibrotic phenotype characteristic of BLF.     

TGF-beta, BMPS, and their signal transducing mediators, Smads, in rat fracture healing

Smads are cytoplasmic signal transducers of transforming growth factor-beta (TGF-beta) and bone morphogenetic proteins (BMPs). Their relation to fracture healing is unknown. This study examined the temporal protein expression of Smads, together with TGF-beta and BMPs, using immunohistochemistry in a rodent fracture model. Over-expression of TGF-beta, BMPs-2, 4, and 7, common-mediator Smad (Smad4), and receptor-regulated Smads (Smads1, 2, 3, and 5) versus lower levels of inhibitory Smad (Smad6), were detected at day 3 in osteogenic cells in the thickened periosteum and bone marrow at the fracture sites. At day 10, Smad6 increased dramatically, Smad2, Smad3, and Smad4 remained elevated while Smad1 and Smad5 decreased in the fracture callus. Smad7 was expressed only in vascular endothelial cells. By day 28, when new bone had replaced the fracture callus, all the protein regulators decreased, approaching control levels. During fracture healing, the expression patterns of Smads1 and 5 were similar to that of BMPs-2 and 7 whereas the expression of Smads2 and 3 was parallel with that of TGF-beta. The Smad family, associated with BMPs and TGF-beta, may play an important role in the early stage of rat fracture healing.     

Inhibition of activin receptor-like kinase 5 attenuates bleomycin-induced pulmonary fibrosis

Activin receptor-like kinase 5 (ALK5) is a type I receptor of transforming growth factor (TGF)-beta. ALK5 inhibition has been reported to attenuate the tissue fibrosis including pulmonary fibrosis, renal fibrosis and liver fibrosis. To elucidate the inhibitory mechanism of ALK5 inhibitor on pulmonary fibrosis in vivo, we performed the histopathological assessment, gene expression analysis of extracellular matrix (ECM) genes and immunohistochemistry including receptor-activated Smads (R-Smads; Smad2/3), CTGF, myofibroblast marker (alpha-smooth muscle actin; aSMA) and type I collagen deposition in the lung using Bleomycin (BLM)-induced pulmonary fibrosis model. ALK5 inhibitor, SB-525334 (10 mg/kg or 30 mg/kg) was orally administered at twice a day. Lungs were isolated 5, 7, 9 and 14 days after BLM treatment. BLM treatment led to significant pulmonary fibrotic changes accompanied by significant upregulation of ECM mRNA expressions, Smad2/3 nuclear translocation, CTGF expression, myofibroblast proliferation and type I collagen deposition. SB-525334 treatment attenuated the histopathological alterations in the lung, and significantly decreased the type I and III procollagen and fibronectin mRNA expression. Immunohistochemistry revealed that SB-525334 treatment showed significant attenuation in Smad2/3 nuclear translocation, decrease in CTGF-expressing cells, myofibroblast proliferation and type I collagen deposition. These results suggest that ALK5 inhibition attenuates R-Smads activation thereby attenuates pulmonary fibrosis.     

Differential expression of osteogenic factors associated with osteoinductivity of human osteosarcoma cell lines

Differential expression of multiple osteogenic factors may be responsible for the different osteoinductivity of osteosarcoma cell lines. We compared in vivo osteoinductivity of human osteosarcoma cell lines (Saos-2 vs. U-2 OS) in nude mice, and their in vitro expression of various osteogenic factors of protein level by quantitative immunocytochemistry and mRNA level by RT-PCR and/or in situ hybridization. Saos-2 cells, but not U-2 OS, were osteoinductive in vivo. Significantly higher expression (independent t-test, all p < 0.005) of osteogenic factors were observed in Saos-2 cells compared with U-2 OS, which included bone morphogenetic proteins (particularly BMPs-2, 3, 4, and 7), transforming growth factor-beta (TGF-beta), BMP receptor (BMPR)-1A, receptor-regulated Smads (R-Smads), Smads 1, 2, and 5, and common-mediator Smad (Co-Smad), Smad 4. In contrast, U-2 OS cells expressed higher levels of inhibitory Smad 6 (I-Smad) protein than Saos-2 cells (p < 0.001). These results suggest that a combination of osteogenic factors (BMPs, TGF-beta, BMPRs, and R/Co-Smads) against I-Smad may play important roles in the Saos-2 cell osteoinductivity. This may have a clinical implication in selecting key osteogenic factors for combined therapy for bone defect diseases. The characterized cell lines can be used as positive and negative controls for the assessments of both in vitro and in vivo bone formation capabilities of designed tissues or biomaterials.     

Smad protein and TGF-beta signaling in vascular smooth muscle cells

Transforming growth factor-beta1 (TGF-beta1) plays a role in vascular remodeling by stimulating vascular smooth muscle cell (SMC) growth and matrix-protein synthesis at sites of vascular injury. Smad proteins have been shown to mediate intracellular signaling of this growth factor. We investigated the expression and phosphorylation of Smads in cultured rat aortic smooth muscle cells. In addition, we evaluated the effects of overexpression of Smad proteins on TGF-beta signal transduction by adenovirus-mediated gene transfer. In rat SMC, Smad1, Smad2, Smad3, Smad4 and Smad5 were detected by immunoprecipitation. Using antisera against phosphorylated Smad2, we showed that TGF-beta1-induced Smad2 phosphorylation in a concentration- and time-dependent manner. Using adenovirus-mediated transfection method, we demonstrated that overexpression of Smad2 or Smad4 was associated with an increased production of TGF-beta1-induced plasminogen activator inhibitor-1 (PAI-1). However, the most prominent expression of PAI-1 was observed upon cotransfection of both Smad2 and Smad4. Both the proliferative effect of TGF-beta1 under serum-free conditions and its anti-proliferative effect under serum-rich conditions were suppressed by the adenovirus-mediated overexpression of Smad7. These results indicated that Smads proteins were expressed in vascular SMC and that they mediated TGF-beta signaling in those cells.     

Mechanisms of tissue inhibitor of metalloproteinase 1 augmentation by IL-13 on TGF-beta 1-stimulated primary human fibroblasts

BACKGROUND: TGF-beta induces expression of tissue inhibitor of metalloproteinase 1 (TIMP-1), a potent inhibitor of matrix metalloproteinases that controls extracellular matrix metabolism and deposition. IL-13 alone does not induce TIMP-1, but in combination with TGF-beta it augments TIMP-1 expression. Although these interactions have implications for remodeling in asthma, little is understood regarding the mechanisms controlling TIMP-1 product. OBJECTIVE: To explore the role of Smads and mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) in the TIMP-1 augmentation by IL-13+TGF-beta1 in primary human airway fibroblasts. METHODS: Real-time PCR, Western blot, ELISA, and transient transfection were used to evaluate the mechanisms of TIMP-1 augmentation. RESULTS: IL-13 enhanced TGF-beta1-induced Smad-2 and Smad-3 phosphorylation, transient transfection with dominant-negative Smad-2 or Smad-3 decreased TIMP-1 mRNA expression in the presence of TGF-beta1 and IL-13+TGF-beta1 through inhibition of Smad-2 or Smad-3 phosphorylation. ERK phosphorylation was increased by IL-13 and IL-13+TGF-beta1. MEK-ERK inhibition decreased TIMP-1 mRNA/protein to a greater degree after IL-13+TGF-beta1 stimulation versus TGF-beta1 alone. MEK-ERK inhibition also significantly increased Akt phosphorylation under all conditions and decreased Smad-3 phosphorylation in the presence of IL-13+TGF-beta1. In contrast, phosphoinositide-3 kinase-Akt inhibition increased phosphorylation of ERK and Smads, leading to increased TIMP-1. CONCLUSION: These results indicate that IL-13 augments TGF-beta1-induced TIMP-1 expression through increased Smad phosphorylation. These increases occur as TGF-beta1 downregulates IL-13-induced phosphoinositide-3 kinase activation while leaving the positive effect of IL-13-induced ERK on Smad signaling. CLINICAL IMPLICATIONS: This augmentation of TGF-beta1-induced TIMP-1 by IL-13 could contribute to the fibrosis and airway remodeling seen in the presence of T(H)2 inflammation in asthma.     

Transforming growth factor-beta- and Activin-Smad signaling pathways are activated at distinct maturation stages of the thymopoeisis

Members of the transforming growth factor (TGF)-beta family play pivotal roles in the control of differentiation, proliferation and tolerance in peripheral T cells. Recently, they have been implicated in thymic selection, but their role is so far not well characterized. In the present study, we demonstrate that specific thymocyte populations are under the influence of either the TGF-beta and/or Activin pathway, and transduce signals into the nucleus via phosphorylated Smad2 (pSmad2). Thymocytes in the medulla and in the subcapsular zone expressed nuclear translocated pSmad2, a hallmark of active TGF-beta/Activin receptor signaling. When analyzed at the cellular level, the pSmad2(+) cells were confined to the double-negative (DN) and single-positive (SP) subpopulations. Moreover, the most immature DN thymocytes (CD44(+)CD25(-) and CD44(+)CD25(+)) expressed higher levels of pSmad2 compared to the more mature DN. In vitro stimulation demonstrated that pure CD44(+)CD25(-), CD44(+)CD25(+) and CD44(+)CD25(+) thymocytes respond to ActivinA, while the mature CD4(+) and CD8(+) SP thymocytes respond to TGF-beta stimulation measured as enhanced phosphorylation of Smad2. Double staining of pSmad2(+) cells with either the Activin type I receptor, ALK4, or the TGF-beta type I receptor, ALK5, demonstrated that pSmad2(+) DN cells exhibited high levels of immunoreactivity to ALK4 and moderate levels of immunoreactivity to the TGF-beta-responsive ALK5 receptor. In sharp contrast, the SP pSmad2(+) cells were predominately ALK5(+). Collectively, our results demonstrate that early and late thymocytes express pSmad2 in the nuclei in vivo. The functional experiments in vitro suggest that members of the TGF-beta family (TGF-beta or Activin) may play important non-redundant roles during different stages of thymopoiesis.     

Expression and localization of the transforming growth factor-beta type I receptor and Smads in preneoplastic lesions during chemical hepatocarcinogenesis in rats

Little is known about the involvement of Smad-related molecules in the regulation of the Transforming Growth Factor (TGF)-beta signaling pathway during hepatocarcinogenesis, particularly with respect to preneoplastic lesions of a rat liver. The aims of this study were to investigate the localizations and temporal expressions of TGF-beta Receptor Type 1 (TGR1) and Smads during the promotion stage of chemical hepatocarcinogenesis in rats. We investigated expressions and localizations of TGR1, Smad2, Smad4, and Smad7 by using semi-quantitative RT-PCR and immunohistochemistry in preneoplastic lesions during rat chemical hepatocarcinogenesis induced by Solt and Farber's method. The down-regulation of TGR1, Smad2, and Smad4 was evident during the later steps of the promotion stage of chemical hepatocarcinogenesis. In contrast with other Smads, increased Smad7 expression was evident during the later steps of the promotion stage. Also immunohistochemistry revealed that the main site of TGR1, Smad2, Smad4, and Smad7 expression was mainly in hepatocytes of the preneoplastic lesions of a rat liver. Dysregulation of the downstream effectors of TGF-beta such as TGR1, Smad2, Smad4 and, Smad7 might contribute to the progression of preneoplastic lesions during chemical hepatocarcinogenesis in a rat.     

Smad3 deficiency ameliorates experimental obliterative bronchiolitis in a heterotopic tracheal transplantation model

Chronic allograft rejection manifested as obliterative bronchiolitis (OB) remains the single greatest impediment to long-term survival after lung transplantation. Transforming growth factor-beta1 (TGF-beta1) has been implicated in the tissue remodeling response associated with OB. Therefore, its intracellular signal transducer, Smad3, is a prime target of investigation. Herein, we examine the role of TGF-beta1, through Smad3, in the development of OB using heterotopic tracheal transplantation in wild-type and Smad3-null mice. TGF-beta1 was detectable within infiltrating mononuclear cells early after transplantation. Later it was detected in fibroblasts and in the connective tissue accumulating within the lumen and the airway wall of the transplanted allografts. Connective tissue growth factor had a similar time and tissue distribution. Nuclear detection of Smad3 and phosphorylated Smads within intraluminal fibroblasts coincided with increased intraluminal deposition of fibronectin and collagen. When transplanted into Smad3-null mice, allografts failed to organize the intraluminal exudates despite fibroblast accumulation and showed reduced fibronectin and collagen deposition. In culture, Smad3-deficient fibroblasts expressed reduced fibronectin in response to TGF-beta1 compared to wild-type cells. Together, these studies suggest that the TGF-beta signal transducer, Smad3, is required for the development of experimental OB in transplanted tracheas.     

Reversible phospho‐Smad3 signalling between tumour suppression and fibrocarcinogenesis in chronic hepatitis B infection

Transforming growth factor (TGF)‐β, type I receptor (TβRI) and c‐Jun N‐terminal kinases (JNK) phosphorylate Smad3 differentially to create 2 isoforms phosphorylated (p) at the COOH‐terminus (C) or at the linker region (L) and regulate hepatocytic fibrocarcinogenesis. This study aimed to compare the differences between how hepatitis B virus (HBV) infection affected hepatocytic Smad3 phosphorylated isoforms before and after anti‐viral therapy. To clarify the relationship between Smad3 phosphorylation and liver disease progression, we studied 10 random patients in each stage of HBV‐related fibrotic liver disease (F1–4) and also 10 patients with HBV‐associated HCC. To examine changes in phosphorylated Smad3 signalling before and after anti‐HBV therapies, we chose 27 patients with chronic hepatitis B who underwent baseline and follow‐up biopsies at 52 weeks from the start of nucleoside analogue treatments (Lamivudine 100 mg daily or Telbivudine 600 mg daily). Fibrosis stage, inflammatory activity and phosphorylated Smad3 positivity in the paired biopsy samples were compared. Hepatocytic pSmad3C signalling shifted to fibrocarcinogenic pSmad3L signalling as the livers progressed from chronic hepatitis B infection to HCC. After nucleoside analogue treatment, serum alanine aminotransferase (ALT) and HBV‐DNA levels in 27 patients with HBV‐related chronic liver diseases were decreased dramatically. Decrease in HBV‐DNA restored pSmad3C signalling in hepatocytes, while eliminating prior fibrocarcinogenic pSmad3L signalling. Oral nucleoside analogue therapies can suppress fibrosis and reduce HCC incidence by successfully reversing phosphorylated Smad3 signalling; even liver disease progressed to cirrhosis in chronic hepatitis B patients.     

Immunohistochemical expression of Smads 1-6 in the 15-day gestation mouse embryo: signaling by BMPs and TGF-betas.

The eight mammalian Smad proteins mediate cellular signaling from members of the transforming growth factor-beta (TGF-beta), bone morphogenetic protein (BMP), and activin families. Smads 1, 5, and 8 transmit signals from BMPs, while Smads 2 and 3 transmit signals from TGF-betas and activin. Smad 4 is a common mediator of both pathways, while Smads 6 and 7 inhibit signaling. Signal transduction involves translocation of Smad complexes to the nucleus and subsequent gene activation. Little is known about the expression of endogenous Smad proteins during development. We identified commercially available Smad antibodies that specifically recognize a unique Smad protein and are suitable for immunohistochemistry. Here we compare the localization of Smads 1, 2, 3, 4, 5, and 6 in tissues of the 15-day gestation mouse embryo. Immunoreactive Smad proteins are seen in many tissues with differences in the localization being dependent upon the cell type. All tissues express Smad 4 and at least one each of the BMP-specific and TGF-beta-specific Smads, while expression of Smad 6 is more restricted. Differences are observed in the nuclear versus cytoplasmic localization among the Smads in different cell types or tissues, suggesting selective activation of Smads during this stage of development.     

Receptor-regulated and inhibitory Smads are critical in regulating transforming growth factor beta-mediated Meckel's cartilage development.

The proper development of Meckel's cartilage is critical for craniofacial skeletogenesis, because it serves as the primordium for the formation of mandible, malleus, incus, and sphenomandibular ligament. Cranial neural crest (CNC) cells contribute significantly to the formation of Meckel's cartilage. Members of the transforming growth factor beta (TGF-beta) family control the proliferation and differentiation of CNC cells during craniofacial skeletogenesis. TGF-beta signaling is transduced from the cell membrane to the nucleus by means of specific type I and type II receptors and phosphorylated Smad proteins. Here we demonstrate that application of TGF-beta promotes chondrogenesis by specifically increasing proliferation of CNC-derived chondrocytes and production of extracellular matrix. To understand the molecular regulation of TGF-beta signaling, we have examined the biological function of both TGF-beta receptor-regulated and inhibitory Smads during Meckel's cartilage development. The expression patterns of Smad2, 3, and 7 are identical to the ones of endogenous TGF-beta and its cognate receptors during Meckel's cartilage development, establishing the potential that these intracellular signaling Smads may regulate TGF-beta-mediated chondrogenesis. Functional haploinsufficiency of Smad2 delays TGF-beta-mediated Meckel's cartilage development. Overproduction of Smad7 severely inhibits Meckel's cartilage formation, indicating a negative feedback on TGF-beta signaling by inhibitory Smad is critical in orchestrating TGF-beta-mediated gene regulation during embryonic chondrogenesis. The effectiveness of TGF-beta signaling is highly sensitive to the level of Smad gene expression.